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Microarray analysis of therapeutic (GM4) and non therapeutic (GM) NOD DC

ABSTRACT: We have previously demonstrated that bone marrow-derived DC can prevent diabetes development and halt progression of insulitis in NOD mice, the mouse model of type 1 diabetes (T1D). The DC population that was most effective in this therapy had a mature phenotype, expressed high levels of costimulatory molecules and secreted low levels of IL-12p70. The protective DC therapy induced regulatory Th2 cells that shifted the dominant Th1 environment, present in NOD mice, to a mixed Th1/Th2 milieu. Microarray analysis of therapeutic and non-therapeutic DC populations revealed several novel molecules that could play important roles in the observed DC-mediated therapy. The therapeutic DC population expressed a unique pattern of costimulatory molecules and chemokines, which were confirmed by flow cytometry and ELISA assays. We have performed in vitro chemotaxis assays that demonstrated the therapeutic DC preferentially attracted Th2 cells, as compared to Th1, Treg or naïve T cells. In addition we quantified the in vivo migration of activated islet-specific T cells to the pancreas using novel cell labeling techniques and 19F nuclear magnetic resonance. A subcutaenous injection of therapeutic DC alters the migration of both Th1 and Th2 cells to the pancreas, and Th1 cells appeared in the lymph node draining the site of DC injection. These results suggest that the therapeutic function of DC is mediated in part by the chemoattractive properties of these DC for diabetogenic Th1 cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE15320 | GEO | 2010/06/24

SECONDARY ACCESSION(S): PRJNA115855

REPOSITORIES: GEO

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Project description:Abstract Two major dendritic cell (DC) subsets have been described in the islets of mice: The immunogenic CD8Î±-CD11b+ DCs and the tolerogenic CD8Î±+CD103+ DCs. We have recently reported on reduced numbers of the minor population of tolerogenic CD8Î±+CD103+ DCs in the pancreas of 5 week old pre-diabetic non-obese diabetic (NOD) mice. Aim: To analyze also the larger subset of CD11c+CD8Î±- DCs isolated from the pancreas of pre-diabetic NOD mice 1) for maturation and tolerance inducing molecules found abnormally expressed on CD8Î±+CD103+ DCs, and 2) for genome-wide gene expression to further elucidate abnormalities in underlying gene expression networks. Methods: CD11c+CD8Î±- DCs were isolated from 5 week old C57BL/6 and NOD pancreas. Expression of cell surface markers including CD86, CCR5, CD11b, CD103, Clec9a, CD24 and CD200R3 were measured by FACS. Genome-wide gene expression by microarray was assessed during the steady state and after in vitro LPS stimulation. Results: The steady state pancreatic CD11c+ CD8Î±- DCs during the pre-diabetic stage showed: 1) A reduced expression of several gene networks important for the prime functions of the cell, such as for cell renewal, immune stimulation and immune tolerance induction, for migration and for the provision of growth factors for beta cell regeneration. This general deficiency state was corroborated by a reduced in vivo proliferation (BrdU incorporation) of the cells and the reduced expression in FACS analysis of CD86, CCR5, CD103, Clec9a, CD24 and CD200R3 on the cells. 2) A hyper reactivity of these cells to LPS correlated with an enhanced pro-inflammatory state characterized by altered expression of a number of classical pro-inflammatory factors and cytokines. Conclusion: The NOD CD11c+CD8Î±- DCs seem to be Janus-faced depending on the conditions: Deficient in steady state with reduced immune stimulation capabilities also for tolerance induction; over-inflammatory with a molecular profile suggesting a preferential stimulatory capacity for Th1 cells when encountering a Pathogen-Associated Molecular Pattern (PAMP) in the form of LPS. We used microarray gene expression analysis to explain the abnormal expression of several cell surface markers involved in tolerace, migration and maturation in the steady-state and to measure the effect of a PAMP such as LPS We isolated RNA from FACS sorted CD11c+CD8Î±- DCs in 10 pooled pancreases from pre-diabetic NOD and non-diabetic C57BL/6 mice at 5 weeks. In addition, we treated in another experiment the isolated pancreas DCs with LPS (and PBS), incubated for 18h and measured gene expression. We compared gene expression between strains NOD vs C57BL/6 under steady-state and after in-vitro LPS/PBS stimulation.

Project description:Gene expression profiling of repeatedly activated compared to recently activated Th1 cells to identify genes that play a role in chronic inflammatory disorders and may qualify as diagnostic or therapeutic targets; ; Upon activation under appropriate costimulatory conditions, naive T helper (Th) cells differentiate into Th2 or Th17 cells, each characterized by the expression of specific effector cytokines. In response to a repeated stimulation with antigen, Th cells develop a stable memory for the expression of those cytokines as well as for other secreted or membrane-associated factors. The stable memory for the expression of proinflammatory effector functions may explain the resistance of Th effector cells to conventional immunosuppressive therapy, and the inability of immunosuppression to cure chronic inflammation. The imprinting of the functional memory is based on epigenetic modifications and expression of distinct transcription factors. In this project, we compare the transcriptomes of once and repeatedly activated murine Th1 cells, to identify genes that induce and maintain the functional memory and control the persistence of pathogenic memory Th1 cells. This in turn might help to discriminate pathogenic versus protective cells in immunopathology and present novel targets for the diagnosis and therapy of chronic inflammatory disease. Experiment Overall Design: Genes differentially expressed in once versus four times stimulated Th1 cells. In vitro polarization of murine naÃ¯ve DO11.10 T cells towards Th1 direction (5 ng/ml recombinant murine IL-12, 5 Î¼g/ml anti-IL-4 antibody) with antigenic stimulation (ova323-339 and irradiated splenic APCs). The transcriptional profiles of resting one week old Th1 (Th1 1w) cells and resting 4 week old Th1 (Th1 4w) cells were compared using Affymetrix Murine Genome (MG) U74V2A GeneChip arrays. Experiment Overall Design: 10 Âµg of total RNA from each cell sample was reverse transcribed using T7-(d)T24 primer and SuperScript II reverse transcriptase Experiment Overall Design: cDNA extraction with a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate Experiment Overall Design: Biotinylated cRNA was transcribed with the MEGAscript high yield transcription kit (Ambion), fragmented, and the hybridization cocktail was prepared (15 Âµg fragmented biotin-labeled cRNA spiked with Eukaryotic Hybridization control) Experiment Overall Design: probes were subsequently hybridised with the GeneChip U74Av2 for 16 hrs at 45 oC Experiment Overall Design: after washing the hybridisation signals were visualised by staining with streptavidin-phycoerythrin and amplification with an anti-streptavidin antibody Experiment Overall Design: TH1_1w_C1; TH1_1w_C2; TH1_1w_C3TH1_4w_C1; TH1_4w_C2; TH1_4w_C3 Experiment Overall Design: 1w: 1 week in culture; 4w: 4 weeks in culture; C1-3: Culture or Experiment No.

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Microarray analysis of therapeutic (GM4) and non therapeutic (GM) NOD DC

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